CN113388635B - Plant double-target-point CRISPR/Cas9 vector and construction method and application thereof - Google Patents

Abstract

The invention discloses a plant double-target CRISPR/Cas9 vector and a construction method and application thereof. First, an intermediate vector with two sgRNA expression cassettes in series is obtained; then pBI121-MCS-Cas9 is obtained; and then a primer containing two sgRNAs is designed, PCR was performed using the intermediate vector as a template; finally, the PCR product was linked into pBI121-MCS-Cas9 by homologous recombination. The plant double-target CRISPR/Cas9 vector can be prepared by the above preparation method, and the pBI121 vector is used as the backbone vector, and the sgRNA expression box 1, the sgRNA expression box 2 and the Cas9 expression box are sequentially connected. The method of the invention improves the construction efficiency of the double-target CRISPR/Cas9 vector, and after obtaining the intermediate vector and the pBI121-MCS-Cas9 vector, it only needs to design a pair of primers for one-step PCR and one-step homologous recombination to be successful It is convenient and quick to construct dual-target CRISPR/Cas9 vectors. In addition, the present invention only needs 20 yuan for one reaction, only needs to synthesize a pair of primers with adapters, and does not require additional primers and other reagents, which significantly reduces the cost.

Description

A plant dual-target CRISPR/Cas9 vector and its construction method and application

technical field

The invention relates to the technical field of molecular biology, in particular to a plant double-target CRISPR/Cas9 vector and a construction method and application thereof.

Background technique

CRISPR/Cas9 has become the most commonly used gene editing system. CRISPR/Cas9 consists of two parts: Cas9 endonuclease and sgRNA (single guide RNA). sgRNA is fused from natural tracrRNA (transactivatingcrRNA) and crRNA (CRISPR RNA). .

The 5' end of the sgRNA includes a 20nt protospacer sequence, which is complementary to the target DNA sequence to achieve double-stranded cleavage; its 3' end is a fixed scaffold sequence with a stem-loop structure, This sequence interacts with the positively charged groove of the Cas9 protein to form a ribonucleoprotein complex (RNP).

At present, the methods for constructing dual-target CRISPR/Cas9 vectors in plants mainly include the following:

(1) Enzymatic ligation-homologous recombination

Taking the construction process of the double-target CRISPR/Cas9 vector in Chinese cabbage as an example, as shown in Figure 1, sgRNA31 and sgRNA32 with adapters are usually designed first, and annealed to form double strands respectively; Ligase joins sgRNA31 and sgRNA32 into its expression cassette (psgR-Cas9-At); then design new primers to clone the sgRNA32 expression cassette, and connect the two sgRNA expression cassettes by homologous recombination; then use Hind III and EcoR I digested the intermediate vector psgR-Cas9-At and the target vector pBI121, and finally connected the two sgRNA and Cas9 expression cassettes into the target vector by T4 DNA ligase, thereby successfully obtaining the target recombinant molecule.

(2) Golden Gate assembly method

The enzymatic ligation of this method can be carried out in the same reaction system and does not need to be carried out in steps. First, use restriction endonuclease to cut the DNA sequence; at the same time, use DNA ligase to connect DNA fragments in a predetermined sequence, and the DNA fragments do not contain restriction sites. In this method, sgRNA expression cassettes (two with restriction sites at both ends) were prepared by cutting and ligating and overlapping extension PCR, and then the sgRNA expression cassettes were assembled into CRISPR/Cas9 vector, so that the target vector was successfully constructed .

As shown in Figure 2, primers containing adapters (OJH307 and OJH308) for Golden Gate cloning were used for amplification using PJG090 as a template. The recommended reagents are as follows: 1 μL PCR product, 50 ng PJG112, 1 μL CutsmartBuffer (NEB), 0.4 μL T4 Ligase Buffer (NEB), 5 U Bsa I (NEB), 20 U T4 DNA Ligase (NEB), and supplement with ddH ₂ O Make up to 10 μL. The reaction was incubated for 20-25 cycles (2 min at 37°C, 5 min at 20°C), followed by incubation at 50°C and 80°C for 5 min, respectively. Subsequently, 1 μL of the product was introduced into Trans T1 competent cells. Positive clones were identified by clonal PCR and sequenced.

(3) Gibson assembly method

This technology requires the addition of homologous fragments at both ends of the DNA fragment by PCR. NEB recommends that the length of the homologous fragment be 15-40 bp, and the corresponding annealing temperature of this part is required to be higher than 48 °C. First, primers need to be designed to amplify the two DNA fragments. At the same time, the two DNA fragments are homologous to each other to facilitate the connection between them; at the same time, the two DNA fragments are homologous to the vector. , to facilitate their connection with the carrier. Then, after the two DNA fragments have been cloned, DNA purification is required. Finally, the linearized vector fragment, these two DNA fragments and Gibsonassembly master mix were incubated for 1 h and directly transformed into competent cells. This method has a very high success rate and generally does not need to screen many clones to get the correct clone. One of the disadvantages of the Gibson assembly cloning method is that it is suitable for the assembly of fragments longer than 200 bp; the second disadvantage is that if the sticky ends form stable secondary structures, such as hairpin structures or stem-loop structures, the success rate will be greatly affected.

(4) Gateway assembly method

Gateway technology is a specific recombination system based on λ phage site (attB×attP→attL×attR). Including two reactions of BP and LR. First, use the BP reaction to create an entry clone, use the recombination reaction between the attB DNA fragment or the expression clone and the attP donor vector, and react at 22°C for 30 min to obtain the entry clone; then perform the LR reaction to obtain one or more In this step, the recombination reaction between the entry clone of attL and the destination vector of attR is carried out at 22°C for 3h to transfer the target sequence in the parallel reaction; to check if the build was successful.

(5) Homologous recombination method

Use online tools to design and synthesize sgRNA (plus adapters), then cut the sgRNA expression cassette with Bbs I enzyme, and connect the sgRNA to its expression cassette (pMD18T-Cas9) by T4 DNA ligase; ) homologous sequence fragment exchange, so that the exogenous DNA fragment replaces the gene in the original site to achieve the purpose of constructing the target vector. Thus, troublesome problems such as lack of regulation and expression due to random insertion and activation or inactivation of genes near the insertion site are avoided.

HD克隆系统将片段AB和CD克隆到pDe-Cas9-D10A或pUC57GW中，流程如图3所示。A schematic of the final gRNA expression cassette is shown in the top panel. Using a set of four universal primers (p1F, p2F, g1R and g2R) and four target-specific primers (g1F and p1R for protospacer target 1, and g2F and p2R for protospacer target 2), Four fragments, A, B, C and D were PCR amplified using the pEn-Chimera-ccdB plasmid in round 1 PCR. In the second round of PCR, primers p1F and g1R were used to fuse fragments A and B to obtain fragment AB, and primers p2F and g2R were used to fuse fragments C and D to obtain fragment CD. In step 3, use

The HD cloning system cloned fragments AB and CD into pDe-Cas9-D10A or pUC57GW, the process is shown in Figure 3.

The construction process of dual-target CRISPR/Cas9 vectors in plants reported in the existing literature is complex, the experimental period is long, and the cost is high. Therefore, a method for constructing dual-target CRISPR/Cas9 vectors with simple construction process, short experimental period and low cost is urgently needed. and carrier.

SUMMARY OF THE INVENTION

In view of the defects in the prior art, in order to simplify the steps of constructing a plant double-target CRISPR/Cas9 vector, improve the efficiency of constructing the vector and reduce the cost of constructing the vector, the present invention discloses a plant double-target CRISPR/Cas9 vector and the same. Construction method and application, first clone the sgRNA expression cassette, insert the sgRNA expression cassette into the psgR-Cas9-At vector through homologous recombination, and obtain an intermediate vector with two sgRNA expression cassettes in series; The frame was inserted into the pBI121 vector to obtain pBI121-Cas9; then the Bpi I restriction site in the sgRNA expression frame was transformed into a multi-cloning site to obtain pBI121-MCS-Cas9; then the primers containing two sgRNAs were designed to connect two sgRNAs in series The intermediate vector of the sgRNA expression cassette was used as a template for PCR; finally, the product of the previous step was linked into pBI121-MCS-Cas9 by homologous recombination. The plant double-target CRISPR/Cas9 vector can be prepared by the above preparation method, and the pBI121 vector is used as the backbone vector, and the sgRNA expression box 1, the sgRNA expression box 2 and the Cas9 expression box are sequentially connected.

The present invention provides a method for constructing a plant double-target CRISPR/Cas9 vector, comprising the following steps:

S1: clone the sgRNA expression cassette, the sgRNA expression cassette is connected with the promoter and the sgRNA scaffold in turn, insert the sgRNA expression cassette into the psgR-Cas9-At backbone vector by the method of homologous recombination, the psgR-Cas9-At backbone The carrier is sequentially connected with a promoter, a sgRNA scaffold, and a Cas9 expression box, the promoter is U6-26 or U3, the nucleotide sequence of the U6-26 is shown in SEQ ID NO. The nucleotide sequence is shown in SEQ ID NO.2, the nucleotide sequence of the sgRNA scaffold is shown in SEQ ID NO.3, and the nucleotide sequence of the Cas9 expression box is shown in SEQ ID NO.4. Step to obtain the intermediate vector of two described sgRNA expression cassettes in series;

S2: insert the sgRNA expression cassette and the Cas9 expression cassette into the pBI121 backbone vector by enzymatic ligation to obtain pBI121-Cas9;

S3: transform the Bpi I restriction site in the sgRNA expression frame in the product of step S2 into a multi-cloning site to obtain pBI121-MCS-Cas9, and the MCS is a multi-cloning site;

S4: According to the DNA sequence of the target gene, design two sgRNA sequences, sgRNA1 and sgRNA2 respectively, add a linker to the sgRNA sequence according to the flanking sequence of the restriction site as a PCR primer, and the PCR primer is obtained in step S1 The described intermediate carrier is a template to carry out PCR amplification;

S5: Connect the PCR product of step S4 into the pBI121-MCS-Cas9 of step S3 by homologous recombination to obtain the plant dual-target CRISPR/Cas9 vector.

Further, the 2 pairs of primers used for cloning the sgRNA expression frame in the step S1 are:

2*U6-26-HR-F1: ACGACGGCCAGTGCCCATTCGGAGTTTTTGTATCTTGTTTC;

2*U6-26-HR-R1: CAAAAACTCCGAATGAAAAAAGCACCGACTCGGTG;

2*U6-26-HR-F2: CCGAGTCGGTGCTTTTTTCATTCGGAGTTTTTGTATCTTGTTTC;

2*U6-26-HR-R2: CAATTTGTGAAATATAAAAAAGCACCGACTCGGTG;

The psgR-Cas9-At backbone vector was linearized with Sma I for single digestion.

Further, in the step S2, the psgR-Cas9-At backbone vector and the pBI121 backbone vector are respectively digested with Hind III and EcoR I double enzymes, and the digested products are recovered by gel cutting after electrophoresis respectively, and then T4 DNA ligase is used. Ligation yields the pBI121-Cas9.

Further, two pairs of primers were designed in the step S3, and the psgR-Cas9-At backbone vector was used as a template to clone the U6-26 promoter and the sgRNA scaffold respectively; Homologous recombination of the pBI121-Cas9 backbone vector, the U6-26 promoter, the sgRNA scaffold and the double-enzyme-cut pBI121-Cas9 backbone vector to obtain the pBI121-MCS-Cas9;

The 2 pairs of primers used in step S3 are:

Bpi-MCS-121HR-F1:CATGATTACGCCCATTCGGAGTTTTTGTATCTTGTTTC;

Bpi-MCS-MR1: CTAAAACAAGCTTGTCGACCTCGAGCAATCACTACTTCGA

CTCTAGCTGTATATAA;

Bpi-MCS-MF2: GTGATTGCTCGAGGTCGACAAGCTTGTTTTAGAGCTAGAA

ATAGCAAGTTAAAATA;

Bpi-MCS-121HR-R2: CAATTTGTGAAATATAAAAAAGCACCGACTCGGTG.

Further, the multiple cloning site in the step S3 is Xho I-Sal I-Hind III.

Further, in the step S5, the pBI121-MCS-Cas9 was linearized by using Xho I and Hind III double enzyme digestion or any one of Xho I, Sal I, and HindIII single enzyme digestion.

The present invention also provides a vector combination for constructing a plant dual-target CRISPR/Cas9 vector, the vector combination is composed of an intermediate vector and pBI121-MCS-Cas9; the intermediate vector uses psgR-Cas9-At as a backbone vector, The psgR-Cas9-At backbone carrier is sequentially connected with a promoter, an sgRNA scaffold, and a Cas9 expression frame, and the intermediate carrier is sequentially connected with a promoter, a sgRNA scaffold, a promoter, an sgRNA scaffold, and a Cas9 expression frame; the pBI121 -MCS-Cas9 uses pBI121 as the backbone vector, and the pBI121-MCS-Cas9 is sequentially connected with a promoter, multiple cloning sites, sgRNA scaffolds, and Cas9 expression cassettes; the multiple cloning sites are Xho I-Sal I-Hind III, the promoter is U6-26 or U3, the nucleotide sequence of U6-26 is shown in SEQ ID NO.1, and the nucleotide sequence of U3 is shown in SEQ ID NO.2, so The nucleotide sequence of the sgRNA scaffold is shown in SEQ ID NO.3, and the nucleotide sequence of the Cas9 expression cassette is shown in SEQ ID NO.4.

The present invention also provides a plant double-target CRISPR/Cas9 vector, the CRISPR/Cas9 vector uses the pBI121 vector as a skeleton vector, and the pBI121 skeleton vector is sequentially connected with sgRNA expression box 1, sgRNA expression box 2, Cas9 expression box frame, the sgRNA expression frame 1 is successively connected with a promoter, sgRNA1, and a sgRNA support, and the sgRNA expression frame 2 is successively connected with a promoter, sgRNA2, and sgRNA support, and the promoter is U6-26 or U3 at the same time, The nucleotide sequence of U6-26 is shown in SEQ ID NO.1, the nucleotide sequence of U3 is shown in SEQ ID NO.2, and the nucleotide sequence of the sgRNA scaffold is shown in SEQ ID NO. 3, the nucleotide sequence of the Cas9 expression cassette is shown in SEQ ID NO.4.

The present invention also provides a method for using a plant double-target CRISPR/Cas9 vector. The double-target CRISPR/Cas9 vector is transformed into Agrobacterium, and then plants are transformed by the flower soaking method or callus, and the obtained T1 generation seeds are treated Screening, extracting the DNA of the positive seedlings for PCR detection, using specific primers to amplify the flanking sequences of the two sgRNAs, and performing electrophoresis detection, sequencing the PCR products with polymorphisms in the electrophoresis strips, and determining the two target genes according to the sequencing results. Whether gene editing has occurred at the sgRNA.

The present invention also provides the application of the construction method of the plant dual-target CRISPR/Cas9 vector in plant gene editing, and the plant is selected from any one of Arabidopsis thaliana, rice, wheat, corn, and sorghum. The U6-26 promoter was selected for Arabidopsis, and the U3 promoter was selected for rice, wheat, maize, and sorghum.

To sum up, compared with the prior art, the present invention achieves the following technical effects:

(1) Improved efficiency: the present invention constructs an intermediate vector containing two tandem sgRNA expression cassettes, and after designing primers, the “sgRNA1-sgRNA scaffold-promoter-sgRNA2” fragment can be obtained by direct one-step PCR reaction, without the need for multiple rounds of PCR Or overlapping PCR and other steps to effectively reduce the experimental steps; the above PCR product and the linearized pBI121-MCS-Cas9 vector can be obtained by one-step homologous recombination to obtain the final vector, which greatly improves the construction of dual-target CRISPR/Cas9 vector efficiency.

(2) Cost reduction: the existing technology uses Golden Gate clone (50 yuan/time), Gibson assembly (150 yuan/time), Gateway clone (125 yuan/time), In-Fusion clone (100 yuan/time), etc. Methods The intermediate carriers or other reagents used in the construction of dual-target CRISPR/Cas9 vectors are relatively expensive, and the construction method of the present invention transforms the Bpi I restriction site in the sgRNA expression frame into a multi-cloning site (Xho I-Sal I -Hind III) to obtain pBI121-MCS-Cas9, Xho I, Sal I and Hind III are much cheaper than Bpi I enzymes; the reagent ClonExpress II One StepCloning Kit used in the present invention only needs 20 yuan for one reaction. In addition, the present invention only needs to synthesize a pair of primers with adapters after the vector combination is successfully constructed, without additional primers and other reagents, and also saves a lot of cost.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Figure 1 shows the process of constructing single-target and dual-target CRISPR/Cas9 vectors by the combination of restriction enzyme ligation and homologous recombination.

Figure 2 shows the process of Golden Gate assembly and construction of a dual-target CRISPR/Cas9 vector.

Figure 3 shows the process of constructing dual-target CRISPR/Cas9 vectors by In-Fusion method.

Fig. 4 is the flow chart of constructing dual-target CRISPR/Cas9 vector according to the present invention.

Figure 5 is a diagram of the psgR-Cas9-At backbone vector of the present invention.

Figure 6 is a diagram of the pBI121 backbone vector of the present invention.

Figure 7 shows the double-target editing results of the Arabidopsis At4g14730 gene.

Detailed ways

In order to make those skilled in the art better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only Embodiments are part of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

The construction method of the plant dual-target CRISPR/Cas9 vector of the present invention is as follows, and the process is shown in Figure 4:

S1: Clone the sgRNA expression cassette, insert the sgRNA expression cassette into the psgR-Cas9-At backbone vector by the method of homologous recombination, and obtain an intermediate vector with two sgRNA expression cassettes in series. The vector diagram of the psgR-Cas9-At backbone vector is shown in the figure As shown in Figure 5;

S2: Insert the sgRNA and Cas9 expression cassettes into the pBI121 backbone vector by the method of enzyme cleavage and ligation to obtain pBI121-Cas9. The vector diagram of the pBI121 backbone vector is shown in Figure 6;

S3: transform the Bpi I restriction site in the sgRNA expression frame into a multi-cloning site to obtain pBI121-MCS-Cas9; the MCS is a multi-cloning site;

S4: Design primers containing two sgRNAs, and perform PCR with the intermediate vector of the two sgRNA expression cassettes in series as a template;

S5: The product of S4 was ligated into pBI121-MCS-Cas9 by homologous recombination.

After the construction of the CRISPR/Cas9 vectors of S1 and S3 of the present invention is completed, in the subsequent application process, only a pair of primers containing two sgRNAs and a linker needs to be designed, and PCR is performed with the intermediate vector of the two sgRNA expression cassettes in series as a template, The product is directly subjected to one-step homologous recombination with the enzyme-cut linearized pBI121-MCS-Cas9 to obtain a dual-target CRISPR/Cas9 vector.

The promoter of the present invention can be selected from U6-26 or U3, U6-26 is suitable for dicotyledonous plants such as Arabidopsis thaliana, and U3 is suitable for monocotyledonous plants such as rice, wheat, corn, sorghum, etc., both of which can realize the dual targets of the present invention CRISPR/Cas9 vector construction. The following examples illustrate the construction method of the present invention by taking U6-26 as an example.

Example 1

(1) construct an intermediate vector containing 2×(pAtU6-26-2×Bpi I-sgRNA scaffold) fragments;

Using CE Design V1.04 software (Nanjing Novizan Biotechnology Co., Ltd.), two pairs of primers were designed to use psgR-Cas9-At as a template to clone the promoter and sgRNA of the sgRNA expression cassette on the psgR-Cas9-At vector respectively. Scaffold, Sma I (ThermoFisher) single-enzyme cut and linearized psgR-Cas9-At vector, homologous recombination of the above two fragments and vector, to obtain an intermediate vector containing 2×(pAtU6-26-2×Bpi I-sgRNA scaffold). The sequences of the two primer pairs used are shown below:

(2) Insert the sgRNA and Cas9 expression cassettes into the pBI121 vector by enzymatic ligation;

The psgR-Cas9-At vector and the pBI121 vector were digested with Hind III (ThermoFisher) and EcoR I (ThermoFisher), respectively, and the digested products were electrophoresed and recovered by gel cutting, and then ligated with T4 DNA ligase to obtain pBI121-Cas9.

(3) transform the Bpi I restriction site in the sgRNA expression frame into a multi-cloning site to obtain pBI121-MCS-Cas9;

Two pairs of primers (table below) were designed, using psgR-Cas9-At as a template to clone pAtU6-26 and sgRNA scaffolds respectively; Hind III and Sma I double digestion pBI121-Cas9 vector, the above two fragments and pBI121-Cas9 were linearized The vector homologous recombination was used to obtain pBI121-MCS-Cas9 using the ClonExpress II One Step Cloning Kit.

(4) Design primers containing two sgRNAs, and perform PCR with the intermediate vector obtained in (1) as a template. PCR system: 2×phanta Max Buffer, 25 μL; dNTP Mix, 1 μL; Bpi-MCS-121HR-F1, 2 μL ; Bpi-MCS-MR1, 2 μL; (1) Intermediate carrier, 1 μL; phanta Max Super-Fidelity DNA Polymerase, 1 μL; ddH ₂ O, 18 μL. PCR program: 94°C, 3 min; 94°C, 30s; 55°C, 30s; 72°C, 90s, 35 cycles; 72°C, 5min.

According to the DNA sequence of the target gene, design 2 sgRNAs, add a linker to the sgRNA sequence according to the flanking sequence of the restriction site, and use the intermediate vector obtained in (1) as a template to carry out PCR with the primer of the linker to obtain "linker- sgRNA1-sgRNA scaffold-pAtU6-26-sgRNA2-linker" fragment.

(5) The product of the previous step was ligated into pBI121-MCS-Cas9 by the method of homologous recombination.

Xho I and Hind III double digestion (or Xho I, Sal I, Hind III single digestion) pBI121-MCS-Cas9 was linearized, the linearized vector and the fragment in (4) were subjected to homologous recombination, using ClonExpress II One StepCloning Kit, you can get the dual target vector "pBI121-pAtU6-26-sgRNA1-sgRNA scaffold-pAtU6-26-sgRNA2-sgRNA scaffold-Cas9".

Example 2

Aiming at the At4g14730 gene of Arabidopsis thaliana, a dual-target CRISPR/Cas9 vector was constructed by using the technology of the present invention, and the Columbia wild-type Arabidopsis thaliana was genetically transformed, and finally an edited plant was obtained, indicating that the method of the present invention is feasible. First, two sgRNAs were designed for At4g14730, namely At4g14730-sg1:GGTGGTAACGAGCTGTATCC and At4g14730-sg2:TCTTTTTCGTCATCCTCCTC. Design and synthesize primers containing adapters, where the bolded parts are the sequence of sgRNA1 and the reverse complement of sgRNA2:

14730-sg1-F: TCGAAGTAGTGATTgGGTGGTAACGAGCTGTATCCGTTTTAGAGCTAGAAATAGCAAGT

14730-sg2-R: TAGCTCTAAAACGAGGAGGATGACGAAAAAGAAATCACTACTTCGACTCTAGCTGT

Using the two steps (4) and (5) in the present invention, a double-target CRISPR/Cas9 vector was successfully constructed, the constructed CRISPR/Cas9 vector was transformed into Agrobacterium, and Arabidopsis thaliana (monocotyledon such as rice) was transformed by the flower soaking method. Plants can be transformed by callus), the obtained T1 generation seeds were screened, the DNA of positive seedlings was extracted for PCR detection, and the flanking sequences of the two sgRNAs were amplified with specific primers 14730SG-FP: GACGGAACAGTAACACCAGC and 14730SG-RP: GCCACAACACTACAACACAC, And electrophoresis detection, found that the electrophoresis strip with polymorphism, take the PCR product for sequencing, the results show that the two sgRNAs of the target gene are edited, as shown in Figure 7, the cleavage site near sgRNA1 is 45 ~ 46bp In between, the cleavage site near sgRNA2 is between 209 and 210 bp, resulting in the deletion of a large fragment of 164 bp.

Based on the above embodiments, in order to simplify the steps of constructing a plant dual-target CRISPR/Cas9 vector, improve the efficiency of constructing the vector and reduce the cost of constructing the vector, the present invention discloses a plant dual-target CRISPR/Cas9 vector and its construction method. Application, first clone the sgRNA expression cassette, insert the sgRNA expression cassette into the psgR-Cas9-At vector through homologous recombination, and obtain an intermediate vector with two sgRNA expression cassettes in series; then insert the sgRNA expression cassette and the Cas9 expression cassette into pBI121 by enzymatic ligation vector to obtain pBI121-Cas9; then transform the Bpi I restriction site in the sgRNA expression box into a multi-cloning site to obtain pBI121-MCS-Cas9; then design primers containing two sgRNAs to connect two sgRNA expression boxes in series The intermediate vector was used as a template for PCR; finally, the product of the previous step was linked into pBI121-MCS-Cas9 by homologous recombination. The plant double-target CRISPR/Cas9 vector can be prepared by the above preparation method, and the pBI121 vector is used as the backbone vector, and the sgRNA expression box 1, the sgRNA expression box 2 and the Cas9 expression box are sequentially connected. The present invention constructs an intermediate vector containing two tandem sgRNA expression boxes, which can directly provide "sgRNA1-sgRNA scaffold-promoter-sgRNA2" fragments for subsequent PCR reactions, reducing experimental steps; The point was transformed into a multi-cloning site (Xho I-Sal I-Hind III) to obtain pBI121-MCS-Cas9, Xho I, Sal I and Hind III are much cheaper than Bpi I enzymes; after transformation, single-enzyme cleavage can be used for linearization The vector can also be linearized by double-enzyme digestion. The latter is more thorough, which is conducive to the next step of homologous recombination reaction; after obtaining the intermediate vector and the pBI121-MCS-Cas9 vector, it is only necessary to design a pair of primers for one-step PCR and One-step homologous recombination can successfully construct a dual-target CRISPR/Cas9 vector, which is convenient and fast.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

sequence listing

SEQ ID NO.1

CATTCGGAGTTTTTGTATCTTGTTTCATAGTTTGTCCCAGGATTAGAATGATTAGGCATCGAACCTTCAAGAATTTGATTGAATAAAACATCTTCATTCTTAAGATATGAAGATAATCTTCAAAAGGCCCCTGGGAATCTGAAAGAAGAGAAGCAGGCCCATTTATATGGGAAAGAACAATAGTATTTCTTATATAGGCCCATTTAAGTTGAAAACAATCTTCAAAAGTCCCACATCGCTTAGATAAGAAAACGAAGCTGAGTTTATACATAG

SEQ ID NO.2

AAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGATGTGAAGAAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGATTTAAAAGAGTTGTGCAGATGATCCGTGGC

SEQ ID NO.3

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC

SEQ ID NO.4

GACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGC TGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAA CGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTAC CCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGA GGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGAC

SEQUENCE LISTING

<110> Jingchu Institute of Technology

<120> A plant double-target CRISPR/Cas9 vector and its construction method and application

<130> 20210701

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 295

<212> DNA

<213> Arabidopsis thaliana

<400> 1

cattcggagt ttttgtatct tgtttcatag tttgtcccag gattagaatg attaggcatc 60

gaaccttcaa gaatttgatt gaataaaaca tcttcattct taagatatga agataatctt 120

caaaaggccc ctgggaatct gaaagaagag aagcaggccc atttatatgg gaaagaacaa 180

tagtatttct tatataggcc catttaagtt gaaaacaatc ttcaaaagtc ccacatcgct 240

tagataagaa aacgaagctg agtttatata cagctagagt cgaagtagtg attgg 295

<210> 2

<211> 380

<212> DNA

<213> Oryza sativa

<400> 2

aaggaatctt taaacatacg aacagatcac ttaaagttct tctgaagcaa cttaaagtta 60

tcaggcatgc atggatcttg gaggaatcag atgtgcagtc agggaccata gcacaagaca 120

ggcgtcttct actggtgcta ccagcaaatg ctggaagccg ggaacactgg gtacgttgga 180

aaccacgtga tgtgaagaag taagataaac tgtaggagaa aagcatttcg tagtgggcca 240

tgaagccttt caggacatgt attgcagtat gggccggccc attacgcaat tggacgacaa 300

caaagactag tattagtacc acctcggcta tccacataga tcaaagctga tttaaaagag 360

ttgtgcagat gatccgtggc 380

<210> 3

<211> 76

<212> DNA

<213> Artificial sequences

<400> 3

gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60

ggcaccgagt cggtgc 76

<210> 4

<211> 4101

<212> DNA

<213> Artificial sequences

<400> 4

gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 60

accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120

agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 180

acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240

ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300

gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360

atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420

ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480

atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540

gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600

aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660

ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 720

attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780

gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840

atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900

ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960

atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020

cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080

tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140

aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200

cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260

attctgcggc ggcaggaaga ttttttaccca ttcctgaagg acaaccggga aaagatcgag 1320

aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380

ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440

gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500

ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560

aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620

ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680

aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740

ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800

aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860

accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920

ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 1980

ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040

ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2100

ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160

gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220

aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280

gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340

aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400

gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460

atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2520

gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2580

aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640

tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700

aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760

gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820

aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880

ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940

caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000

cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060

atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120

atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3180

ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3240

accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3300

acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3360

agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3420

tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3480

gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3540

ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3600

tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3660

aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3720

tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3780

cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3840

ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 3900

atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 3960

gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4020

gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4080

ctgtctcagc tgggaggcga c 4101

Claims (8)

1. A construction method of a plant double-target-point CRISPR/Cas9 vector is characterized by comprising the following steps:

s1: cloning a sgRNA expression frame, wherein the sgRNA expression frame is sequentially connected with a promoter and a sgRNA scaffold, the sgRNA expression frame is inserted into a psgR-Cas9-At framework vector by a homologous recombination method, the psgR-Cas9-At framework vector is sequentially connected with a promoter, a sgRNA scaffold and a Cas9 expression frame, the promoter is U6-26 or U3, the nucleotide sequence of U6-26 is shown as SEQ ID NO.1, the nucleotide sequence of U3 is shown as SEQ ID NO.2, the nucleotide sequence of the sgRNA scaffold is shown as SEQ ID NO.3, the nucleotide sequence of the Cas9 expression frame is shown as SEQ ID NO.4, and an intermediate vector for connecting two sgRNA expression frames in series is obtained;

s2: inserting the sgRNA expression frame and the Cas9 expression frame into a pBI121 skeleton vector by an enzyme digestion connection method to obtain pBI121-Cas 9;

s3: reconstructing a Bpi I enzyme cutting site in the sgRNA expression cassette in the product of the step S2 into a multiple cloning site to obtain pBI121-MCS-Cas9, wherein the MCS is the multiple cloning site;

s4: designing 2 sgRNA sequences according to the DNA sequence of a target gene, namely sgRNA1 and sgRNA2, adding a joint on the sgRNA sequence according to a flanking sequence of an enzyme cutting site to serve as a PCR primer, and carrying out PCR amplification by taking the intermediate vector obtained in the step S1 as a template by using the PCR primer;

s5: and (3) connecting the PCR product obtained in the step S4 into the pBI121-MCS-Cas9 obtained in the step S3 by a homologous recombination method to obtain the plant double-target CRISPR/Cas9 vector.

2. The method for constructing the sgRNA expression cassette of claim 1, wherein the 2 pairs of primers used for cloning the sgRNA expression cassette in step S1 are:

2*U6-26-HR-F1：ACGACGGCCAGTGCCCATTCGGAGTTTTTGTATCTTGTTTC；

2*U6-26-HR-R1：CAAAAACTCCGAATGAAAAAAGCACCGACTCGGTG；

2*U6-26-HR-F2：CCGAGTCGGTGCTTTTTTCATTCGGAGTTTTTGTATCTTGTTTC；

2*U6-26-HR-R2：CAATTTGTGAAATATAAAAAAGCACCGACTCGGTG；

the psgR-Cas9-At scaffold vector was linearized with a single enzyme cut using Sma I.

3. The construction method according to claim 1, wherein in step S2, HindIII and EcoRI are used to double-cleave the psgR-Cas9-At skeleton vector and the pBI121 skeleton vector respectively, the cleavage products are separated by electrophoresis, and then recovered by gel cutting, and then connected by T4DNA ligase to obtain the pBI121-Cas 9.

4. The construction method according to claim 1, wherein two pairs of primers are designed in step S3, and the U6-26 promoter and the sgRNA scaffold are cloned respectively by using the psgR-Cas9-At framework vector as a template; HindIII and Sma I are subjected to double enzyme digestion on the pBI121-Cas9 skeleton vector, the U6-26 promoter, the sgRNA bracket and the pBI121-Cas9 skeleton vector subjected to double enzyme digestion are subjected to homologous recombination to obtain the pBI121-MCS-Cas 9;

the 2 pairs of primers used in step S3 were:

Bpi-MCS-121HR-F1：CATGATTACGCCCATTCGGAGTTTTTGTATCTTGTTTC；

Bpi-MCS-MR1：CTAAAACAAGCTTGTCGACCTCGAGCAATCACTACTTCGA

CTCTAGCTGTATATAA；

Bpi-MCS-MF2：GTGATTGCTCGAGGTCGACAAGCTTGTTTTAGAGCTAGAA

ATAGCAAGTTAAAATA；

Bpi-MCS-121HR-R2：CAATTTGTGAAATATAAAAAAGCACCGACTCGGTG。

5. the method of claim 1, wherein the multiple cloning site in step S3 is Xho I-Sal I-Hind III.

6. The method according to claim 1, wherein step S5 is performed by using Xho I and Hind III double digestion or Xho I, Sal I, and Hind III single digestion to linearize pBI121-MCS-Cas 9.

7. A vector combination for constructing a plant dual-target CRISPR/Cas9 vector, which consists of an intermediate vector and pBI121-MCS-Cas 9; the middle vector takes psgR-Cas9-At as a skeleton vector, a promoter, a sgRNA bracket and a Cas9 expression frame are sequentially connected to the psgR-Cas9-At skeleton vector, and the promoter, the sgRNA bracket, the promoter, the sgRNA bracket and the Cas9 expression frame are sequentially connected to the middle vector; the pBI121-MCS-Cas9 takes pBI121 as a skeleton vector, and a promoter, a multiple cloning site, a sgRNA bracket and a Cas9 expression frame are sequentially connected to the pBI121-MCS-Cas 9; the multiple cloning site is Xho I-Sal I-Hind III, the promoter is U6-26 or U3, the nucleotide sequence of U6-26 is shown as SEQ ID No.1, the nucleotide sequence of U3 is shown as SEQ ID No.2, the nucleotide sequence of the sgRNA scaffold is shown as SEQ ID No.3, and the nucleotide sequence of the Cas9 expression cassette is shown as SEQ ID No. 4.

8. The application of the construction method of the plant double-target point CRISPR/Cas9 vector disclosed by claim 1 in plant gene editing, wherein the plant is selected from any one of Arabidopsis, rice, wheat, corn and sorghum.

Images